Presently, there is a general burden need for fast, sensitive and quantitative diagnostic technologies. Such ones are suitable for wide market areas including public health, research, farming, environmental care, veterinary medicine, and certain industrial production areas. Improved sensitivity, speed, robustness, stability, and decreased cost per analysis are factors, which after being accomplished in diagnostic technologies can find applications in quite new areas.
Very high sensitivity can be obtained with certain diagnostics instruments, but they are too expensive. On the other hand, certain methods can be enough inexpensive, exemplified by immunochromatography, but they are not applicable to fully quantitative applications of the market. Any technology, wherein a set of satisfactory demands are met, will have an important place in the future diagnostics and a huge market potential.
There are a number of different analytical principles in practical use in diagnostics: for example, assays based on radioactivity, enzyme-linked immunosorbent assay (ELISA), colorimetric assays, and assays based on fluorescence, and chemiluminescence including anodic as well as hot electron-induced (cathodic) electrochemiluminescence (ECL). The hot electron-induced ECL is described in detail in U.S. Pat. No. 6,251,690, Kulmala S., et al. Each of these techniques has their role as regards to the integral of sensitivity, robustness, stability, speed, and price. The differences between the techniques reflect the function of physical limitations or advantages of the methods. For example, a drawback of the application based on radioactive compound is the decay of the label within a period of time and the extra cost of radioactive waste from both the safety and environmental viewpoint. The application of the most sensitive assays on diagnostics is limited by the complicated nature of the tests and instruments, and only experts can perform the assays. The complexity of the assay is generally directly proportional to the price of the instrument and/or the test. In the context of complex instruments, it could be mentioned the anodic electrochemiluminescence techniques now becoming more and more popular: the instrument is a complicated laboratory robot, the handling of which needs expertise and where the measuring process involves repeated washes and preparative steps. They are factors that increase the cost of the analyses as well as increase the amount of waste and therefore will make this method impossible for the needs of small laboratories, doctors offices etc. (bedside or point of care analytics).
Commercially beneficial methods are based on the principle that the substances to be analyzed are identified and measured in mixtures by so-called label substances. In the measurements based on unique properties of biological molecules, as in immunochemical assays, the analyte to be measured (X) can be selectively sorbed from a mixture of molecules to solid-phase bound antibodies and then the bound molecules are measured with another labeled antibody selectively binding to (X). The label substances can be radioactive isotopes, enzymes, light absorbing, fluorescent or phosphorescent molecules, certain metal chelates etc., which are linked covalently to the antibody. Alternatively, the purified (X) can be marked and the amount of unknown unlabeled sample (X) can be measured by a competition reaction. The assays for DNA and RNA can be also based on the selective binding (bioaffinity). Also many other chemical and biochemical analyses can be carried out by the same principles. In order to decrease the cost and/or increase the measuring accuracy, there is presently a tendency to measure several different parameters at the same time in the sample. One possibility is to use labels fluoresceing or phosphoresceing (luminating) at different wavelength or possessing different fluorescence lifetimes. Different measuring principles and strategies, which can be used in immunodiagnostics, have been described in the book The Immunoassay Handbook, Edited by David Wild, Stockton Press Ltd., New York, 1994, on pages 1-618.
It is known in the prior art that organic substances and metal chelates are beneficial as label substances and that they can be excited by light or by electrochemically to produce luminescence specific to the label. These methods are particularly sensitive and feasible. However, because the measured concentrations are extremely low, there are also case-dependent difficulties; the use of fluorescence can be disturbed, among other things, by Tyndall, Rayleigh and Raman scattering. When measuring biological substances, there is, almost without exception, after the excitation pulse, a fast-discharging high background fluorescence. Phosphorescence in the solution phase can be utilized mostly only with chelates between lanthanide ions and specially synthesized organic molecules. The drawback of the excitation techniques with the photoluminescent labels is the complexity of the instruments and the high price of the sensitive optical components.
In general, the advantage of ECL is the low price of the electrical excitation components and simpler optics. Thus, compared to the photoluminescence, several drawbacks can be avoided. Traditional anodic electrochemiluminescence with inert metal electrodes can be carried out with organic luminophores by a relative simple instrument in non-aqueous solvents. However, in bioaffinity assays, where the biggest commercial expectations are concentrated to, water solutions are applied. Biological samples are taken nearly always in non-organic solutions and therefore the measuring system should work in aqueous or at least in micellar water solutions. Only a very limited number of transition metal chelates are working as ECL-labels in anodic ECL in water or micellar solutions.
Thus far the commercially most important analytical chemical application of the anodic ECL is the method using derivatives of Ru(bpy)32+-chelate, where the detection phase of the label occurs in micellar phase. As known from textbooks, the micellar mixtures are always prone to different disturbing effects due to the uncontrolled complexity of the micellar equilibria. Thus, the hot electron-induced ECL, which does not depend on micelles has many crucial advantages over the anodic ECL. It can be applied both to immuno- and DNA hybridization methods (see, Blackburn, G., et al., 1991, Clin. Chem. 37: 1534-1539; Kenten, J., et al. 1992, Clin. Chem. 33: 873-879). The immunoassays and DNA or RNA probe applications by Roche Diagnostics Ltd. exploit magnetic particles by which the label substance is brought onto golden working electrode (Massey; Richard J., et al. U.S. Pat. No. 5,746,974; Leland; Jonathan K., et al. U.S. Pat. No. 5,705,402). The reproducible handling of magnetic latex particles is however in many ways difficult, therefore this method is useful only in expensive laboratory robots (e.g. Elecsys 1010 and 2010) having a complicated and precise liquid handling system. In addition, the permanent massive golden work electrode needs long cleaning and pretreatment between each analysis (Elecsys Service Manual, p. 70).
It was found out that a significant improvement in the performance could be acquired with placing a thin porous film on the work electrode, and by producing CIPF-devices (see patent US2009178924, Ala-Kleme et al.). However, these kind CIPF devices had a serious drawback in that they need a separate counter electrode in the measurement instrument and careful washing steps between each analysis because the same electrolytic cell containing the counter electrode has to be repeatedly used.
In HECL work electrode, the hot electrons are considered to tunnel from the closest parts of the electrode from the counter electrode, which means electrode edges in case of planar electrodes in the same plain because theoretically the tunneling of electrons shall happen from places which has highest tunneling probability (see Kulmala S., Suomi J, Analytica Chimica Acta 500 (2003) 21-69). If the tunneling will take place from such edges and even if a detectable luminescence will occur, such a local luminescence would not be useful as the work electrode aimed at the diagnostics purposes of the present invention because such luminescence would not be dependent on the concentration of the label compounds bound to the surface of the electrode, or at least, the relationship would be only marginal. Thus, a person skilled in the art would not try to construct electrodes wherein the work and counter electrodes are fabricated on approximately same level on a flat surface if HECL is applied. If the electrons are tunneled from sharp edges, it follows that it is extremely difficult to control the manufacturing process so that the different electrodes would function reproducible, the fact what is absolutely necessary for diagnostic test chips.
In the present invention, we surprisingly found that it is possible to construct even completely planar integral work and counter electrodes which have a very high performance in bioaffinity assays. This appeared to be result from materials used in the electrodes and the proper dimensions of the thickness of the electrolyte solution above the electrodes as well geometrical arrangements of the electrodes themselves. This finding led also to a completely new property of the electrodes which enable further increase of the electrode performance over the present situation. We surprisingly found that the described construction with certain electrode materials allowed the use of bipolar pulses for luminescence excitation, meaning that cathode and anode can be repeatedly changed as to their position either repeatedly or after a certain pulse trains. This allows exploiting the surface of both electrodes for the analysis purposes. For example, the light pulses from one electrode can be compared to the other one and such a system serves as an internal standard, or two analytes can me measured simultaneously.
According to then present invention a significant quantitative and qualitative improvement of the ECL electrodes was achieved as illustrated in the patent claims 1-10.